Lithium reagent composition, and method and device for determining lithium ion amount using same

ABSTRACT

To provide to a reagent composition used in quantitative measurement of lithium in aqueous solutions such as biological specimens and environmental liquid samples by a simple colorimeter or ultraviolet-visible light spectrophotometer immediately, a reagent composition which permits to measure a concentration of lithium by visual observation, and a method and apparatus for measuring lithium ion by using the reagent. A reagent composition for measuring lithium comprising F28 tetraphenylporphyrin compound as chelating agent, including further a water-soluble organic solvent, a pH modifier and a stabilizer, and a method and apparatus for measuring lithium ion by using the reagent.

This is a continuation of U.S. application Ser. No. 14/387,444, filedDec. 1, 2014, which is a National Stage of International Application No.PCT/JP2012/061015, filed Apr. 25, 2012, claiming priority based onJapanese Patent Application No. 2012-087928, filed Apr. 6, 2012; thecontents of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

This invention relates to a reagent composition used in quantitativemeasurement of lithium in an aqueous solution such as biologicalspecimens and environmental liquid samples, and to method and device fordetermining the quantity of lithium ions by using the reagentcomposition.

BACKGROUND ART

It is known that lithium-containing drugs are effective in feelingstabilization and anti-depression, so that they are used widely asfeeling-stabilizer and antidepressant drugs. Tablets of lithiumcarbonate (for oral administration) are generally prescribed as afeeling stabilizer as well as a drug for bipolar disorder (circulatorypsychosis) or anti-depressive drug.

However, when such lithium-containing drug is administrated to patients,it is necessary to control or adjust the concentration of lithium inserum within a proper range. In fact, the lithium carbonate (Li₂CO₃) hassuch a characteristic that its administration effect is exhibited onlywhen the concentration of lithium in blood arrives at nearly a “lithiumpoisoning level”. In other words, when the drug is administrated, thetherapeutic drug monitoring (TDM) is indispensable so as to monitor thelithium concentration in blood, since a therapeutic range is very nearto the poison level.

In practice, it is necessary to control or limit the concentration oflithium in a patient blood sample within a limited range of generallyfrom 0.6 to 1.2 mEq/L. In fact, when the lithium concentration in serumis lower than 0.6 mEq/L, no anti-depressive effect is expected. On thecontrary, excess administration over 1.5 mEq/L of the concentration inplasma will result in the lithium poisoning. Overdose result in a fatalcause of symptoms of poisoning including tremor, alalia, nystagmus,renal disturbance and convulsion. Therefore, when a sign of latentlydangerous symptoms of lithium-poisoning is observed, treatment with suchlithium-containing drug must be stopped and the concentration in plasmamust be re-measured so as to take a necessary measurement and to easethe lithium-poisoning.

Thus, the lithium salt is an effective medicine in the treatment ofdepressive patients, but overdose result in serious troubles. Therefore,when a lithium-containing anti-depressive drug is administered, it isindispensable to monitor the concentration of lithium in serum and toassure that the concentration is always kept with a limited range offrom 0.6 to 1.2 mEq/L. Therefore, the quantitative measurement oflithium in serum is necessary in the treatment of depression patient.

Several liquid reagent compositions that permit colorimetricdetermination of lithium for the clinical laboratory test have beendeveloped.

-   Patent Document 1 discloses a reagent composition used to measure    the concentration of lithium in a biological sample by using primary    color body cryptideinofa.-   Patent Document 2 discloses an analytical reagent which reacts with    lithium ion, comprising a macrocyclic compound having a pyrrole ring    and eight bromine (Br) atoms combined at β position of the pyrrole    ring.-   Non-Patent Document 1 discloses that lithium ion can be detected by    a compound in which all hydrogen bonded to carbons of    tetraphenylporphyrin are replaced by fluorine.

LIST OF PRIOR ARTS

-   Patent Document 1 JP-A1-7-113807-   Patent Document 2 EP 1283986-B1-   Non-Patent Document 1 Analytical Chemistry Vol. 51, No. 9, pp.    803-807 (2002); K. Koyanagi et al., “Synthesis of F28    tetraphenylporphyrin and its use for separation and detection”

SUMMARY OF INVENTION Problems to be Solved by the Invention

Known lithium reagent compositions, however, have such demerits orproblems that they are poisonous compositions, that drug substances areexpensive or are not supplied stably, and that most drug substances donot dissolve in water or, even soluble, deactivated in water, so thatcoloring reaction is very slow.

Above-mentioned non-Patent Document 1 was developed to solve the aboveproblems and permits use of color developing technique. The method ofthis non-Patent Document 1, however, requires a dilution operation of aspecimen since the sensibility is too high and the specification of thelithium reagent composition requires a range of over pH 11, so that itis easily deteriorated with CO₂ in air and hence measured data are notstable. Still more, no concentrated aqueous solution other than those ofsodium hydroxide and of potassium hydroxide for a range of over pH11 isavailable in practice uses, so that it is difficult to keep a constantconcentration. These concentrated aqueous solutions are hazardoussubstances which are difficult to handle so that use of which should beavoided. Their storage requires special containers and a larger scalespecial equipment or installation is required in their handling.Therefore, this technology is difficult to apply to on-site monitoringand POCT (Point Of Care Testing).

The reagent composition for measuring the quantity of lithium disclosedin Patent Document 1 is completely different from the present inventionand can be used only at pH 12. As stated above, in a range of over pH11, there is no concentrated aqueous solution in practice other thanthose of sodium hydroxide and of potassium hydroxide which is hazardoussubstances which are difficult to be handled and a larger scale specialequipment or installation is required for their supplement.

The document of Koyanagi et al., of the non-Patent Document 1 teachesthat lithium ion can be separated and detected by using F28tetraphenylporphyrin. However, extraction with oily poisonous chloroformis necessary to perform the separation and detection of lithium ion. Infact, direct determination of lithium in aqueous solution withoutcomplicated pretreatment was impossible.

Thus, there was a problem that rapid and quantitative measurement oflithium ion in serum was impossible. In fact, detection of lithium ionin aqueous solutions by using F28 tetraphenylporphyrin is not easy sothat quantitative measurement of lithium ion with this compound have notbeen realized until now.

This invention was made to solve the problem and provides a reagentcomposition used in quantitative measurement of lithium (concentration)in aqueous solutions such as biological specimens and environmentalliquid samples, and to a measuring method and device using the reagentcomposition for determining the quantity of lithium ion. This inventionpermits to measure the concentration of lithium rapidly or immediatelyby using the conventional colorimeter. This invention provides also alithium reagent composition which can be used for screening by visualobservation and method and apparatus using the lithium reagentcomposition to measure lithium ion.

Means to Solve the Problems

A subject of this invention is a reagent composition for lithium(“lithium reagent composition” hereafter), characterized in that itcomprises a compound having a structure represented by the formula (I):

in which all hydrogens bonded to carbons of a tetraphenylporphyrin arereplaced by fluorine, a water-soluble organic solvent and a pH modifier.

Lithium in an aqueous solution such as a biological specimen and anenvironmental sample generates a color with the lithium reagentcomposition according to the present invention, in particular with theabove compound in which all hydrogens bonded to carbons of atetraphenylporphyrin are replaced by fluorine, which functions as achelating reagent (color developer).

Color change from yellow to red by a coloring reaction which is observedbetween a F28 tetraphenylporphyrin compound and lithium ions isdifficult to be realized. However, what is requested is to determineprecisely a quantity of lithium in serum in the range of 0.6 mg/dL to2.0 mg/dL (0.9 mM to 3 mM). Inventors found such a fact that thequantity of lithium in serum can be determined precisely by setting aconcentration of the F28 tetraphenylporphyrin compound in a range of 0.1to 1.0 g/L, preferably 0.5 g/L in an embodiment of this invention.

The pH modifier is used preferably in the present invention. In fact, inan acidic side lower than pH 5.0, the F28 tetraphenylporphyrin compoundwhich is a color developer (chelating reagent) according to thisinvention does not bond to lithium ion, so that no coloration change isobserved and it is difficult to determine the quantity of lithium. In arange between pH 5 and pH 7, a specific reaction occurs between thecolor developer and lithium ion but the coloring reaction speed is slow.In a range between pH 8 and pH 11, the color developer reacts withlithium ion rapidly and a stable coloring complex can be formed. Inalkaline side of higher than pH 11, a color tone of the chelatingreagent and of coloring complex formed becomes instable in time. Thismay be caused by absorption of carbon dioxide in air, so that pHfluctuates. Therefore, it is necessary to use a pH modifier or pH bufferthat can keep pH of the lithium reagent composition according to thepresent invention in a range from pH 7 to pH 12, preferably from pH 8 topH 11.

The pH modifier can be selected from alkali medicine including sodiumhydroxide, potassium hydroxide and ammonia, acid medicine includingacetic acid, phosphoric acid, citric acid, carbonic acid, bicarbonicacid, oxalic acid, hydrochloric acid, nitric acid and their salts. ThepH modifier may be pH buffer and may be selected from citric acid,carbonic acid, bicarbonic acid, phosphoric acid, succinic acid, phthalicacid, ammonium chloride, sodium hydroxide, potassium hydroxide, MES asGood's buffer, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES,DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO,CAPS and their salts.

The lithium reagent composition according to the present inventionpermits the specific color reaction for lithium in a range of from pH 5to pH 12 by incorporating the pH modifier.

It is indispensable that the solvent (polar solvent) used in thisinvention is an organic solvent that is compatible with water. Thesolvent can be a solution consisting mainly of organic solvent or anaqueous solution in which an organic solvent is added, provided that thesolvent can be mixed uniformly with an aqueous solution such as serum,blood plasma and eluate which is a test sample. In fact, since a testsample to be measured is in a form of an aqueous solution when theconcentration of lithium in sample is determined by a general-purposetype automated analyzer and by an ultraviolet-visible lightspectrophotometer, it is desirable that the reagent composition is in aform of an aqueous solution.

The organic solvent is preferably chosen from dimethylsulfoxide (DMSO),dimethylformamide (DMF) and dimethylacetamide (DMA).

In actual products, a suitable stabilizer is incorporated in the reagentcomposition according to this invention. In an embodiment, a surfactantis used as the stabilizer. The surfactant improves the dispersibility ofF28 tetraphenylporphyrin compound and prevents suspensions originatedfrom the sample during the coloring reaction. Therefore, the stabilizeris used to assure such effect.

The stabilizer may be nonionic surfactant or anionic surfactant. Thenonionic surfactant may be sorbitan fatty acid ester, pentaerythritolfatty acid part ester, propylene glycol monofatty acid ester, glycerinfatty acid monoester, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylenefatty acid part ester, polyoxyethylene sorbitol fatty acid part ester,polyoxyethylene fatty acid ester, fatty acid di-ethanol amide, fattyacid ethanol amide, polyoxyethylene fatty acid amide, polyoxyethyleneoctylphenyl ether (Triton X-100 ®), p-nonyl phenoxy polyglycidol ortheir salts. Preferable nonionic surfactants are polyoxyethyleneoctylphenyl ether (Triton X-100 ®) and p-nonyl phenoxy polyglycidol.

The anionic surfactant as stabilizer may be alkyl sulfate ester salt,polyoxyethylene alkyl ether sulfate salt, polyoxyethylene phenyl ethersulfate salt, alkyl benzene sulfonate and alkane sulfonate. Typicalanionic surfactant is selected from sodium dodecyl sulfate, sodiumdodecyl benzene sulfonate and sodium polyoxyethylene alkylphenyl ethersulfate.

The lithium reagent composition according to the this invention cancontain more than one masking reagent, in order to avoid disturbancecaused by other ions than lithium, which may present in the sample, tosuppress oxidation of the reagent composition and to improve the storagestability. The masking reagent may be not necessary if there are fewions other than lithium.

The masking reagent which can be added to the lithium reagentcomposition according to the present invention may be selected fromtriethanolamine, ethylenediamine,N,N,N′,N′-tetrakis(2-pyridylmethylethylenediamine (TPEN), pyridine,2,2-bipyridine, propylene diamine, dimethylene triamine, dimethylenetriamine-N,N,N′,N″,N″-penta acetic acid (DTPA), trimethylene tetramine,trimethylene tetramine-N,N,N′,N″,N′″,N′″-hexaacetic acid (TTHA),1,10-phenanthroline, ethylene diamine tetraacetic acid (EDTA),O,O′-bis(2-aminophenyl)ethyleneglycol-N,N′,N′-tetraacetic acid (BAPTA),N,N-bis(2-hydroxyethyl)glycine (Bicine),trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CyDTA),O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (EGTA),N-(2-hydroxyl)imino diacetic acid (HIDA), imino diacetic acid (IDA),nitrile triacetic acid (NTA), nitrile trimethylphosphonate (NTPO) andtheir salts. Triethanol amine is preferably used.

The lithium reagent composition according to this invention may includeantiseptics to prevent degradation caused by microorganism. Theantiseptics are not limited especially and may be sodium azide andProcline®. An amount of antiseptics is not especially limited and may bea concentration used generally as an antiseptic. For example, in case ofsodium azide, the amount of antiseptics is about 0.1% by mass to areaction solution. The antiseptics are usually prescribed for productswhich are stored for longer term duration.

To guarantee a long-term storage, the lithium reagent compositionaccording to the present invention can be stored separately in a form ofa kit for measuring lithium reagent comprising two separate reagentswhich are mixed just before measurement to realize the lithium reagentcomposition of claim 1. For example, a first reagent comprises thestabilizer and the pH modifier or pH buffer, while a second reagentcomprises the tetraphenylporphyrin compound, water-miscible organicsolvent, stabilizer and pH modifier or pH buffer.

In actual uses, the lithium reagent composition according to the presentinvention is contacted with a test sample of serum and/or blood plasmato induce coloring of the lithium complex which is measured in term ofabsorbance and spectrum so as to determine a quantity of lithium in thesample by comparing with reference concentrations of a standard samplewhose lithium concentrations are of known.

In practice, in the coloring of the lithium complex and in its spectrum,the sensitivity is measured preferably at a wavelength of 550 nm or inthe vicinity of wavelength from 530 nm to 560 nm, or the sensitivity ismeasured at a wavelength of 570 nm or in the vicinity of wavelength from565 nm to 650 nm to calculate the concentration of lithium. In thiscase, the sensitivity is understood as the absorbance or a difference inabsorbance in an ultraviolet-visible light spectrophotometer.

In the measuring device, the coloring, absorbance or spectrum of thelithium complex generated from the lithium reagent composition accordingto the present invention contacted with a test sample of serum and bloodplasma is measured, or the sensitivity at a wavelength of 550 nm or inthe vicinity of wavelength from 530 nm to 560 nm or the sensitivity at awavelength of 570 nm or in the vicinity of wavelength from 565 nm to 650nm is measured to calculate the quantitative value of lithium.

Advantages of Invention

The lithium reagent composition according to the present invention andthe method and device for measuring lithium ions permit to determine ormeasure the concentration of lithium in an aqueous solution such asenvironmental sample and biological specimen easily. In the lithiumreagent composition defined in claims 1 to 13, the calibration curve ofthe concentration of lithium is linear in a practical range of from 0.6to 1.2 mEq/L, so that the concentration can be calculated by a simpleoperation from numerical values of the colorimeter and of theultraviolet-visible light spectrophotometer. Therefore, the lithiumconcentration in serum sample or biological specimen can be determinedquickly and quantitatively by usual spectrophotometer. The resultingdata can be used as a management index in TDM treatment for example. Or,the quantitative determination of a larger number of specimens can bedone in a short time by an automatic analyzer for clinical chemistry.

In the present invention, the lithium reagent composition is adjusted toa pH range of from pH 5 within pH 12 so as to enable measurement by thespectrometry. In an acidic range of under pH 5, the chelating reagentaccording to the present invention (F28 tetraphenylporphyrin lithium)does not bond to helium ions so that change in color which is dependenton the lithium concentration is not observed. On the contrary, in analkaline side of over pH 12, a color tone of the chelating reagent andof coloring complex formed is not stable. The stability of the colortone becomes poor due to absorption of carbon dioxide in air which is acause of pH fluctuation. In the pH range from pH 5 to pH 7, the specificcoloring of the chelating reagent can be observed since the chelatingreagent bonds to lithium ions but the coloring speed is too slow.Therefore, the pH range from pH 8 to pH 11 is preferable, since, in thepH range from pH8 to pH11, the chelating reagent bonds to lithium ionrapidly and coloring reaction is specific and stable.

Metal complex of tetraphenylporphyrin possesses a typical specificspectrum range in the vicinity from 380 nm to 460 nm called the “Soretband” in which the maximum sensitivity is obtained. This range may beselected as a measuring wavelength range. However, the sensitivity inthis range is too high for a lithium concentration having clinicalsignificance in a serum sample, so that dilution operation is necessary,resulting in increase of complicated operations and of additional unitsfor dilution, which increase a size of measuring unit.

In the present invention, a wavelength of 550 nm or in the vicinityrange of from 530 nm to 560 nm in which the sensitivity is lower byseveral times than that of the Soret band is used as the measuringwavelength range. By selecting this range, the optimum sensitivity isobtained for a concentration of sample to be tested and complicateddilution operation and dilution unit can be eliminated. Still more, thecalibration curve according to the present invention has betterlinearity than that of in case of the Soret band, so that theconcentration can be calculated easily from the measured values obtainedby a small size colorimeter or an ultraviolet visible lightspectrophotometer. Still more, change in color tone from yellow to redis very sharp in the present invention, the level of concentration canbe judged by visual observation or naked eyes.

If the Soret band is used as a photometry wavelength, there is suchanother problem that the quantitative value of lithium is influenced byother organic substances and color components such as nitrate ion,creatinine, bilirubin, biliverdine and hemolyses hemoglobin. Thisinfluence or problem can be reduced in the present invention and theconcentration of lithium can be determined with high precision.

In the conventional method for measuring lithium, a large scale singlepurpose apparatus was required. In this invention, the concentration oflithium can be determined by a small portable colorimeter and can beconstructed as a POCT kit.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A table for calculating the optimum concentration of F28tetraporphyrin according to this invention

FIG. 2 Graphs of ultraviolet-visible light spectrophotometer obtained inthe result of Example 1 according to this invention.

FIG. 3 Graph of the calibration curve at different wavelengths inExample 1 according to this invention.

FIG. 4 Graphs showing change in spectrum (color reaction) when F28tetraphenylporphyrin-lithium complex is formed in Example 1 according tothis invention.

FIG. 5 A graph showing a correlation between measured values of serumsamples in Example 1 according to this invention and measured valuesobtained by the atomic absorption method (conventional method).

FIG. 6 [Table 1] showing a comparison with measured values obtained byusing an automated analyzer in which the control serum samples wereused.

FIG. 7 Table 2 showing how to detect lithium by visual observation inthis invention.

FIG. 8 A graph of an absorbance spectrum in Example 1 according to thisinvention.

FIG. 9 [Table 3] showing measured values obtained by different organicsolvents according to this invention.

FIG. 10 [Table 4] showing measured values obtained by differentstabilizers according to this invention.

FIG. 11 [Table 5] showing measured values obtained by different maskingreagents according to this invention.

MODE FOR CARRYING OUT THE INVENTION

Inventors studied lithium reagent compositions which can be used formeasuring a concentration of lithium in serum and blood plasmaquantitatively and more simply and focused on a compound represented bythe general formula (I):

in which all of hydrogen atoms bonded to carbons of atetraphenylporphyrin ring are replaced by fluorine atoms (the totalnumber of fluorine is 28) in the macro cyclic compound disclosed innon-Patent Document 1 and complete the present invention. The abovecompound is called herein “F28 tetraphenylporphyrin”.

Patent Documents 2 and 3 disclose similar lithium reagent compositionscomprising a macro cyclic compound having pyrrole rings in which eightbromine atoms (Br) are boned to β position of the pyrrole ring, toprovide an analytical reagent which can react with lithium ions. Thiscompound, however, is difficult to react with lithium if pH is not in analkali side above pH 11.

In case of the F28 tetraphenylporphyrin according to the presentinvention, the reaction occurs in a range of pH 5 to pH 12. In thepresent invention, the F28 tetraphenylporphyrin is used as a chelatingreagent and is used to determine the lithium ions in an aqueous systemquantitatively.

Now, the lithium reagent composition according to the present inventionis explained in much in details by using Examples.

EXAMPLES Example 1 Sample 1

In this Example 1, a first reagent as a pH buffer solution and a secondreagent as a coloring reagent solution were prepared firstly. Then, tworeagents of the first and second reagents were mixed just beforemeasuring operation to prepare a lithium reagent composition accordingto the present invention. Although these two reagents can be stored in aform of mixer but it is advisable to store them separately and mixtogether just before measuring operation to avoid deterioration of thereagents during a long storage time duration.

Now, we will explain how to prepare the reagent composition according tothe present invention in details.

To begin with, the first reagent (pH buffer solution) is prepared.Followings are the composition of the first reagent.

-   (1) First reagent (as stabilizer and buffer solution):    -   chelating reagent: none    -   organic solvent; none    -   stabilizer (dispersant: nonionic surfactant): 1.0% by weight of    -   TritonX-100 ® (polyoxyethylene octylphenyl ether)    -   masking reagent: 10 mM of triethanol amine

Into a mixture of above components, 7% by weight of ammonium chloridewas added to adjust to pH 10. Then, the total volume was increased to 1liter with purified water and stored in a usual storing container. If aproportion of TritonX-100 ® (polyoxyethylene octylphenyl ether) is lowerthan 1.0% by weight, turbidity may occur in some cases. On the contrary,if excess stabilizer is used, foam will be generated in a reactorvessel. Such turbidity or forming may influence the reproducibility ofmeasurement, so that a range of range of 0.1 to 5.0% by weight ispreferable and 1.0% by weight is more preferable.

In this Example, the masking reagent is 10 mM of triethanol amine. If anamount of the masking reagent is short, a satisfactory masking effectwill not be obtained in such samples that contain excess foreign ionsother than lithium. On the contrary, excess masking reagent will masklithium ion itself, resulting in a cause of errors in measurement.Therefore, a range of 1.0 to 100 mM is preferable and 10 mM is morepreferable.

The second reagent (color developing reagent solution) is produced asfollows.

-   (2) Second reagent (as color developing reagent solution):    -   chelating reagent: 0.5 g/L of F28 tetraphenylporphyrin    -   organic solvent; 20% by weight of dimethylsulfoxide (DMSO)    -   stabilizer (dispersant: nonionic surfactant): 1.0% by weight of        TritonX-100 ® (polyoxyethylene octylphenyl ether)    -   masking reagent: 10 mM of triethanolamine

Into a mixture of above components, 0.05M (mol/L) of MOPS (Good'sbuffer) was added to adjust to pH 7.0. Then, the total volume wasincreased to 1 liter with purified water and the resulting solution wasstored in a usual storing container.

In Example 1, color development reaction of F28 tetraphenylporphyrincompound is difficult. However, in the practical clinical laboratorytest for measuring the concentration of lithium in serum, the accuracyin a lithium concentration range of 0.6 mM to 3 mM is required. Inventorfound that the precise measurement can be done by selecting theconcentration of F28 tetraphenylporphyrin compound to 0.1 to 1.0 g/L,preferably 0.5 g/L.

In the concentration range of lithium of 0.6 mM to 3 mM, measurement oflithium can be performed advantageously by setting the concentration ofF28 tetraphenylporphyrin compound in the final reagent composition to0.1 to 1.0 g/L, preferably 0.5 g/L. If the concentration is lower thanthe above limit, a reaction between F28 tetraphenylporphyrin and lithiumion is not sufficiently proceed. On the contrary, if the concentrationexceeds the above limit, another trouble of increase in the absorbanceof a blank of F28 tetraphenylporphyrin compound will occur. Therefore,the concentration of 0.5 g/L is preferably used.

In more details, the reaction between F28 tetraphenylporphyrin andlithium ion is a reaction of equal mole ratio of 1:1 to form a chelatecomplex. When a test sample containing 3 mM of lithium is reacted withthe reagent composition according to the present invention under thecondition of Example 1, the concentration of lithium in the reactionsystem becomes 0.02 mM. Therefore, the concentration of F28tetraporphyrin compound must exist at a concentration of higher than0.02 mM to effect the reaction sufficiently (neither too much nor toolittle).

In the complex-forming reaction (coloring reaction) between a chelatingreagent and metal ions, it is necessary in general to use the chelatingreagent (F28 tetraporphyrin) at an amount of from equal mol to ten timesmols with respect to a reactant or a subject to be tested (lithium). Asis shown in FIG. 1 which shows the optimum concentrations of F28tetraporphyrin, the reagent composition is prepared in such a mannerthat the concentration of F28 tetraporphyrin during the reaction timebecomes from equal mol to 10 times. In practice, it is preferable to usea concentration of the chelating reagent in the reagent composition of0.5 g/L (5 times) rather than 0.1 g/L (same size) so as to permit to usein wider measuring conditions, because parameters of dosages atmeasuring reaction of an added amount of the reagent composition and ofan amount of sample to be tested depend on measuring apparatus anddesired thresholds and vary. For example, in case of a measuringapparatus whose measuring accuracy is not so high, an amount of samplemay be increased to two times to five times to that of Example 1. Toprepare to such cases, it is advisable to use the concentration of 0.5g/L (5 times) of the reagent composition which is enough amount ofreagent for the reaction. Excess amount of higher than 10 times has noadvantage because increased amount of reagent may not significantadvantage in the kinetic of coloring reaction but rather increase atrouble of elevation of blank level.

What is necessary is to satisfy the reaction condition in the mole ratiobetween chelating reagent and lithium. For example, when theconcentration of chelating reagent (F28 tetraporphyrin) in the secondreagent is 1.0 g/L, an amount of the second reagent which is added tothe reaction can be reduced to a half. Or, when an amount of sample isreduced to a half, an amount of the chelating reagent can be reduced toa half.

In Example 1, the concentration of F28 tetraphenylporphyrin is 0.5 g/L.The optimum concentration of F28 tetraphenylporphyrin is 0.1 to 1.0 g/Lthat satisfies the reaction condition in mole and lowers to the minimumblank level.

An amount of dimethylsulfoxide (DMSO) is 5 to 30% by weight. When thisamount is shorter, dispersion of F28 tetraphenylporphyrin in a solutionbecome poor. On the contrary, if excess amount of dimethylsulfoxideresult in increase of the organic solvent in the reagent composition.Therefore, a preferable amount is 20% by weight.

F28 tetraphenylporphyrin used in this Example 1 has a structurerepresented by the following formula (I):

in which all hydrogens bonded to carbons of a tetraphenylporphyrin arereplaced by fluorine atoms.

-   (3) Now, we will explain how to prepare a calibration curve of the    lithium reagent composition prepared by mixing the first reagent    with the second reagent for samples whose lithium concentrations are    known.

In Example 1, 720 μL of the first reagent (buffer solution) and 240 μLof the second reagent (coloring reagent solution) were added to 6 μL ofa sample. In this case, the first reagent has a buffer capacity at pH10.After the first and second reagents and the sample are mixed, theresulting mixture of a test liquid has about pH 10.

Thus, when F28 tetraphenylporphyrin according to the present inventionis used as a chelating reagent, color developing reaction can be carriedout in a pH range of from pH 5 to pH 10. In fact, the present inventionprovides a reagent for lithium measurement possessing a strong pHbuffering action in a range of lower than pH 10, so that fluctuation ofpH caused by absorption of CO₂ in air can be reduced. And hence, anadverse effect to measured values can be avoided, and it is possible tostore the measuring reagents in general-purpose containers.

It is possible to mix the first reagent with the second reagent justbefore usage and to add the resulting mixture to the same volume ofsample. In this case, 940 μL of the liquid mixture can be added to 6 μLof a sample.

A test sample was added to the resulting mixture of pH 10 to effect areaction at ambient temperature for 10 minutes and then an absorbance at550 nm was measured by a ultraviolet-visible light spectrophotometer(HITACHI, U-3900 type), the blank being the test sample. FIG. 2 showsthe result which is a relation between absorbance and Li concentration(mg/L). FIG. 4 is a graph showing change in spectrum in a visible lightrange when F28 tetraphenylporphyrin-lithium complex is formed.

For metal complex of tetraphenylporphyrin, the maximum sensitivity isobtained at a wavelength range of so-called Soret band (about from 380nm to 460 nm). However, in the present invention, this Soret band rangeis not used but a wavelength of 550 nm or in the vicinity range of from530 nm to 560 nm is used, so that complicated operations of dilution anddilution means or an auxiliary facility are not necessary in the presentinvention.

FIG. 3 showing graphs of the calibration curves at different wavelengthsreveals that better linearity in the calibration curve can be obtainedwhen a wavelength of 550 nm or in the vicinity range of from 530 nm to560 nm is used than cases when wavelengths of so-called Soret band areused. Therefore, the precise concentration can be calculated easily by asimple colorimeter or spectrophotometer. Still more, change in colorfrom yellow to red is very sharp, so that a level of the concentrationcan be detected easily by naked eyes. In the conventional technique, anapparatus of a large scale for exclusive use is necessary to measure thelithium concentration, while, in the present invention, the lithiumconcentration can be measured easily by a portable colorimeter orultraviolet-visual light spectrophotometer which is used widely. Thepresent invention can be constructed in a form of a POCT kit.

In the graph of FIG. 3, a line (●) was obtained in a wavelength of 550nm which was used in Example 1, while other two carves were obtained inwavelengths of 405 nm (*) and 415 nm (x) that corresponds to wavelengthsof Soret band when the same procedure as Example 1 was repeated. In thecases of 405 nm (*) and 415 nm (x), however, measurement was carried outafter the samples were diluted at 5 times since the sensitivity was toohigh. FIG. 3 reveals that a calibration curve having a good linearitycan be obtained for the wavelength of 550 nm of Example 1, but thecalibration curves of the wavelengths of 405 nm and 415 nm are notlinear.

FIG. 4 shows changes in spectrum when F28 tetraphenylporphyrin-lithiumcomplex is formed. It is confirmed clearly from FIG. 4 that theabsorbance will increase linearly with the increase of lithiumconcentration from 6 mg/dL to 1.2 mg/dL, 1.8 mg/dL, 2.4 mg/dL and 3.0mg/dL. An absorption peak of 415 nm (Soret band) which is typical forporphyrin-metal complex and an absorption peak of 550 nm (shown in FIG.4) increase and an absorption peak of 570 nm (also shown in FIG. 4)decreases in proportion to the concentration of lithium. Therefore, adifference in absorbance can be calculated in these absorption peaks. Inthe present invention, the wavelength of 550 nm is preferably used as aphotometry measuring wavelength because of good linearity in thecalibration curve.

It is possible to select a wavelength range from 540 nm to 560 nm as thephotometry measuring range in place of the wavelength of 550 nm used inExample 1. In fact, some measuring equipment may not have a photometryfilter for 550 nm. In such case, the photometry measuring wavelength canbe selected from a wavelength range in the vicinity such as 540 nm or560 nm where the sensitivity is also high. A wavelength of 570 nm alsocan be used as a photometry measuring wavelength, since decrease in thesensitivity of absorbance at 570 nm is also quantitative as is shown inFIG. 4. Therefore, a difference in absorbance (Δ Abs) at 570 nm also canbe calculated with a reference of the reagent as a blank.

In such a rare case that some contaminants that interfere at thewavelength of 550 nm are produced in a sample of patient and erroneousdata are produced at the wavelength of 550 nm, it is possible to selecta wavelength of 570 nm or in the vicinity of from 565 nm to 650 nm asphotometry measuring wavelength to avoid such trouble and to calculatethe lithium concentration from a decrease in the sensitivity as adifference in absorbance.

Now, we will explain experimental data of Example 1 which show that thelithium concentration can be measured at high accuracy with the lithiumreagent composition of according to the present invention.

Results of Experiment by Ultraviolet-Visible Light Spectrophotometer(HITACHI, U-3900 Model)

FIG. 2 shows an experiment result measured by an ultraviolet-visiblelight spectrophotometer (HITACHI, U-3900 model). An axis of abscissa isknown lithium ion concentrations (Li concentration, mg/dL) and an axisof ordinate is difference in absorbance measured by theultraviolet-visible light spectrophotometer at a wavelength of 550 nm.

FIG. 2 reveals that a good linearity is obtained in a relation betweenthe absorbance and the lithium concentration.

Correlation Test Between Atomic Absorption Method (Conventional Method)and the Method According to this Invention for a Serum Sample

FIG. 5 is a graph showing a correlation of measured values between themeasuring method of Example 1 according to this invention and theconventional atomic absorption method (conventional method) carried outfor the same serum sample. Measured values obtained by the conventionalatomic absorption method (conventional method) are plotted on axis ofabscissa (X), while measured values according to this invention areplotted on axis of ordinate (Y). A regression line shown in FIG. 5 showsa good correlation of more than 95%. This result reveals that lithium ina serum sample can be determined quantitatively by anultraviolet-visible light absorptiometry with the reagent compositionaccording to the present invention.

Comparison of Measured Values Carried Out by Automatic Analysis forControl Serum Samples

The lithium concentration was measured for following control serumssamples in which the lithium concentration is valued:

-   -   Precinorm U (Roche)    -   Precipath U (Roche)    -   Pathonorm H (SERO AS)    -   Auto norm (SERO AS)        by using a biochemistry automated analyzer (HITACHI, H-7700        model) at a photometry measuring wavelength of 546 nm (which is        a wavelength set in this analyzer and is near to 550 nm) by 1        point end method.        Device Parameters:    -   Reagent: 0.24 mL    -   Sample: 0.005 mL    -   Photometry wavelength (main/sub): 546 nm/700 nm    -   Measuring time: 10 minutes    -   Temperature: 37° C.    -   1 point end: increasing method

Results shown in [Table 1] of FIG. 6 proves such a fact that thatmeasured values obtained by the present invention coincide with theguaranteed values under the above conditions, so that it was confirmedthat the lithium concentration in serums can be measured satisfactorilyby an automated analyzer for clinical tests.

Detection of Lithium by Visual Observation

[Table 2] of FIG. 7 shows results of visual observation for test sampleliquids. In this test, 920 μL of a coloring reagent solution which was amixture of the first reagent and the second reagent according to thepresent invention was added to 8 μL of a test sample and the resultingmixture was reacted for 10 minutes at ambient temperature before thevisual observation was effected. Developed colors were compared with acolor tone guide prepared by using control serums in a form of thestandard lithium concentration solutions at different concentrationlevel of lithium.

Clear change in color from yellow to red was confirmed in respectiveconcentration levels and the change in color of the control serumscoincide with the color guide of the control serums. From this fact, itwas proved or confirmed that the lithium concentration in serum can bedetermined quickly and easily without using specific equipment accordingto the present invention.

As explained above, it is confirmed that the lithium concentration canbe measured at high accurately by using the lithium reagent of Example 1according to the present invention.

Example 2

Procedure of Example 1 was repeated but the first reagent in the lithiumreagent composition was changed by adding 0.1 M (mol/L) of MOPS toadjust to pH 8.0 and by adding pure water up to the total volume of 1liter. Namely, a mixture of the first reagent, the second reagent andthe reagent was adjusted to nearly pH=8 at a measuring time.

-   (1) The first reagent (as stabilizer and buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant nonionic surfactant): Triton X-100 @        polyoxyethylene octylphenyl ether: 1.0% by weight    -   Masking agent: triethanol amine: 10 mM,        0.1M of MOPS was added to the above mixture to adjust pH of the        mixture to pH 8. Then, the total volume was increased to 1 liter        with purified water and the resulting solution was stored in a        general purpose storing container.-   (2) The second reagent (as a coloring reagent solution):    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: Dimethylsulfoxide (DMSO): 20% by weight    -   Stabilizer (dispersant, nonionic surfactant): TritonX-100 @        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Masking agent: triethanolamine 10 mM        To a mixture of above components, 0.05M of MOPS (buffer) was        added to adjust pH to pH 7.0 and purified water was added up to        the total volume of 1 liter, which was stored in a        general-purpose container.

In the same manner as Example 1, 720 μL of the first reagent (buffersolution) of and 240 μL of the second reagent (coloring reagentsolution) were added to 6 μL of a test sample at a time when the lithiumconcentration was measured. After the reaction was continued for 10minutes at ambient temperature, the absorbency was measured at 550 nmwavelength by an ultraviolet-visible light spectrophotometer (HITACHIU-3900 type)

Experimental Result in the Ultraviolet-Visible Light Spectrophotometer(HITACHI U-3900 type)

FIG. 8 is a graph of the experimental result of the ultraviolet-visiblelight spectrophotometer (HITACHI, U-3900 type). The abscissa (X) isknown lithium ion concentrations (Li concentration, mg/dL) and theordinate (Y) plots differences in the absorbance at 550 nm in theultraviolet-visible light spectrophotometer.

FIG. 8 reveals that the difference in absorbance is dependentlyproportional to the lithium concentration for the reagent compositionprepared at pH 8 or under a measurement condition of pH 8 and that agood linearity of a calibration curve is obtained at pH 8 also.

However, at the measurement condition of pH 8, the reaction kinetics alittle slows down and is quantitatively stabilized in about 10 minute to20 minutes. In case of pH 10, the reaction completes within 10 minutes.Therefore, in the buffer system in a range of pH 5 to pH 10 of lithiumreagent composition of this invention, there is no necessity to use abuffer system of above pH 11 based on a thick hydroxide solution such assodium hydroxide and potassium hydroxide in case, and hence handlingoperation becomes simpler. The pH range can be set according to desiredneeds and is adjusted preferably to pH 10 in which the reaction kineticsis rapid and sufficient buffer power can be maintained with Good'sbuffer, ammonium chloride system and carbonic acid system. From apractical point of view, it is advisable to carry out with a pH 10buffer system of Example 1 in which the reaction advances accurately.

Thus, in the lithium reagent composition according to this invention, itis necessary to use a pH buffer which functions as pH modifier foradjusting pH to a range from 7 to 12 or a pH buffer as pH modifier. Moredesirably, it is preferable to use a pH modifier or pH buffer whichadjusts pH to pH 8 to pH 11, and more preferably to use a pH modifier orpH buffer which adjust pH around pH 10.

Example 3

Now, selection of the organic solvent will be explained. In theinvention, it is important that the solvent is an organic solvent whichis miscible with water since the reaction solutions to be measured areaqueous solutions such as serum. The solvent can be a liquid consistingmainly of an organic solvent or an aqueous solution containing anorganic solvent, provided that the components in the reagent compositionare stabilized as an aqueous solution. In particular, when the lithiumconcentration in the sample is measured by a general-purpose automatedanalyzer and by an ultraviolet-visible light spectrophotometer, it isdesirable to use basically an aqueous solution containing organicsolvent.

Other organic solvents which can be mix with water than Examples 1, 2are explained in Example 3. In Example 3, the same procedure as Example1 was repeated but the organic solvent of the second reagent ofdimethylsulfoxide (DMSO) (20% by weight) in the lithium reagentcomposition was replaced by dimethylformamide (DMF) (20% by weight).

-   (1) The first reagent (as buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant: nonionic surfactant): TritonX-100 ®        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Making reagent: triethanolamine 10 mM,        Into a mixture of above components, 7% by weight of ammonium        chloride was added to adjust pH to pH 10 and purified water was        added up to the total volume of 1 liter, which was stored in a        general-purpose container.-   (2) The second reagent (as coloring reagent solution)    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: Dimethylformamide (DMF): 20% by weight    -   Stabilizer (dispersing agent: nonionic surfactant) TritonX-100®        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Masking reagent: triethanolamine 10 mM        Into a mixture of above components, 0.05M of MOPS (buffer) was        added to adjust pH to pH 7.0 and purified water was added up to        the total volume of 1 liter, which was stored in a        general-purpose container.

Example 4

As the organic solvent which is miscible with water, dimethylsulfoxide(DMSO) (20% by weight) was used in Example 1 and dimethylformamide (DMF)(20% by weight) was used in Example 2.

In this Example 4, a lithium reagent composition was prepared by usingdimethylacetamide (DMA) (20% by weight) as an organic solvent which ismiscible with water and the concentration of lithium was measured by thelithium reagent composition.

-   (1) The first reagent (as buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant: nonionic surfactant): TritonX-100 ®        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Making reagent: triethanolamine 10 mM,        Into a mixture of above components, 7% by weight of ammonium        chloride was added to adjust pH to pH 10 and purified water was        added up to the total volume of 1 liter, which was stored in a        general-purpose container.-   (2) The second reagent (as coloring reagent solution)    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: dimethylacetamide (DMA): 20% by weight    -   Stabilizer (dispersing agent: nonionic surfactant) TritonX-100 ®        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Masking reagent: triethanolamine 10 mM        Into a mixture of above components, 0.05M of MOPS (buffer) was        added to adjust pH to pH 7.0 and purified water was added up to        the total volume of 1 liter, which was stored in a        general-purpose container.

FIG. 9 shows the results of a comparison of lithium detected in thecontrol serum sample, in which the concentration of lithium was measuredby the same procedure as Example 1 but the organic solvent was changedfrom Dimethylsulfoxide (DMSO) of Example 1 to dimethylformamide (DMF) inExample 3 and to dimethylacetamide (DMA) in Example 4. [Table 3] of FIG.9 shows results of a comparison between the conventional measuringmethod and the measuring method according to the present invention.

[Table 3] of FIG. 9 showing “Comparison among different organicsolvents” shows following results: a measured value obtained by usingdimethylformamide (DMF)(20% by weight) as organic solvent which ismiscible with water in Example 1 was 0.83 mM (mmol/L); a measured valueobtained by using dimethylformamide (DMF) (20% by weight) as organicsolvent which is miscible with water in Example 3 was 0.82 mM (mmol/L);and a measured value obtained by using dimethylacetamide (DMA) (20% byweight) as organic solvent which is miscible with water in Example 4 was0.81 mM (mmol/L). These values coincide over 95% with a measured valueobtained by atomic absorption spectrophotometry 0.82 mM (mmol/L).Therefore, it is possible to determine quantitatively and accurately theconcentration of lithium in aqueous samples such as serum by dispersingF28 tetraphenylporphyrin uniformly in these organic solvents to preparethe liquid reagent composition according to the present invention.

Example 5

In this Example 5, selection of stabilizer for the lithium reagentcomposition according to the present invention is explained.

Use of stabilizers for the lithium reagent compositions in Examples 5 to7 is basically same as Example 1 but the stabilizer was changed tononionic surfactant alone (Example 5), anionic surfactant (Example 6)and both of nonionic surfactant and anionic surfactant (Example 7)respectively.

In following Example 5, the lithium reagent composition contains onlynonionic surfactant (TritonX-100®)) (polyoxyethylene octylphenyl ether)as the stabilizer. Other components in the lithium reagent compositionare same as Example 1.

Example 5

-   (1) The first reagent (as buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant: nonionic surfactant): TritonX-100 ®        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Making reagent: triethanolamine 10 mM        Into a mixture of above components, 7% by weight of ammonium        chloride was added to adjust pH to pH 10 and purified water was        added up to the total volume of 1 liter, which was stored in a        general-purpose container.-   (2) The second reagent (as coloring reagent solution)    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: Dimethylsulfoxide (DMSO): 20% by weight    -   Stabilizer (dispersing agent: nonionic surfactant) TritonX-100®        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Masking reagent: triethanolamine 10 mM        Into a mixture of above components, 0.05M of MOPS (buffer) was        added to adjust pH to pH 7.0 and purified water was added up to        the total volume of 1 liter, which was stored in a        general-purpose container.

Example 6

In Example 6, the composition contains only anionic surfactant (sodiumdodecyl sulfate (Wako Junyaku).

-   (1) The first reagent (as buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant: anionic surfactant only):        -   sodium dodecyl sulfate (Wako Junyaku) 1.0% by weight    -   Making reagent: triethanolamine 10 mM,        Into a mixture of above components, 7% by weight of ammonium        chloride was added to adjust pH to pH 10 and purified water was        added up to the total volume of 1 liter, which was stored in a        general-purpose container.-   (2) The second reagent (as coloring reagent solution)    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: Dimethylsulfoxide (DMSO): 20% by weight    -   Stabilizer (dispersing agent: anionic surfactant only) (sodium        dodecyl sulfate (Wako Junyaku) 1.0% by weigh    -   Masking reagent: triethanolamine 10 mM        Into a mixture of above components, 0.05M of MOPS (buffer) was        added to adjust pH to pH 7.0. Then, the total volume was        increased to 1 liter with purified water and the resulting        solution was stored in a general-purpose storing container.

Example 7

In Example 7, the composition contains both of anionic surfactant and ofnonionic surfactant as stabilizer in the lithium reagent composition.

-   (1) The first reagent (as buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant: nonionic surfactant and anionic        surfactant):        -   (a) nonionic surfactant: TritonX-100® (polyoxyethylene            octylphenyl ether) 1.0% by weight        -   (b) anionic surfactant: sodium dodecyl sulfate (Wako            Junyaku) 1.0% by weigh    -   Making reagent: triethanolamine 10 mM,        Into a mixture of above components, 7% by weight of ammonium        chloride was added to adjust pH to pH10. Then, the total volume        was increased to 1 liter with purified water and the resulting        solution was stored in a general-purpose storing container.-   (2) The second reagent (as coloring reagent solution)    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: Dimethylsulfoxide (DMSO): 20% by weight    -   Stabilizer (dispersing agent: nonionic surfactant and anionic        surfactant)        -   (a) nonionic surfactant: TritonX-100® (polyoxyethylene            octylphenyl ether) 1.0% by weight        -   (b) anionic surfactant: sodium dodecyl sulfate (Wako            Junyaku) 1.0% by weigh    -   Masking reagent: triethanolamine 10 mM        Into a mixture of above components, 0.05M of MOPS (buffer) was        added to adjust pH to pH 7.0. Then, the total volume was        increased to 1 liter with purified water and the resulting        solution was stored in a general-purpose storing container.

The concentration of lithium in the control serum sample was determinedquantitatively by the same procedure as Example 1 by using lithiumreagent compositions prepared in Example 5, Example 6 and Example 7.Results are summarized in [Table 4] of FIG. 10 “Comparison of measuredvalues among different stabilizers”.

FIG. 10 reveals that measured values coincide over 95% among a measuredvalue for the nonionic surfactant alone (0.82 mM), a measured value foranionic surfactant alone (0.82 mM) and a measured value for twosurfactants (0.83 mM).

This result shows that almost same measured values can be obtainedregardless of surfactant type used or their combination. Therefore, thesurfactants can be used in combined for a sample in which suspension orturbidity is worried about.

Now, selection of the masking reagent for the lithium reagentcomposition is explained. In above-mentioned Examples, triethanolaminewas used as a masking reagent for the lithium reagent composition, butethylenediamine tetra acetic acid (EDTA) also can be used.

Example 5 shows a case of a lithium reagent composition containingtriethanolamine as masking reagent, Example 8 shows a case ofethylenediamine tetraacetic acid (EDTA) alone and Example 9 shows a casecontaining both masking reagents.

Example 8

In Example 8, potassium ethylenediamine tetraacetic acid (EDTA, 2K)alone was used as a masking reagent.

-   (1) The first reagent (as buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant: nonionic surfactant): TritonX-100 ®        (polyoxyethylene octylphenyl ether) 1.0% by weight    -   Making reagent: ethylenediamine tetra acetic acid (EDTA 2K)        (Dojin Chemical) 10 mM        Into a mixture of above components, 7% by weight of ammonium        chloride was added to adjust pH to pH10 and purified water was        added up to the total volume of 1 liter, which was stored in a        general-purpose container.-   (2) The second reagent (as coloring reagent solution)    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: Dimethylsulfoxide (DMSO): 20% by weight    -   Stabilizer: TritonX-100® (polyoxyethylene octylphenyl ether):        1.0% by weight    -   Masking reagent: ethylenediamine tetra acetic acid (EDTA 2K)        (Dojin Chemical): 10 mM        Into a mixture of above components, 0.05M of MOPS (buffer) was        added to adjust pH to pH 7.0. Then, the total volume was        increased to 1 liter with purified water and the resulting        solution was stored in a general-purpose storing container.

Example 9

In Example 9, both of triethanolamine and ethylenediamine tetraaceticacid (EDTA 2K) are used in combination as a masking reagent.

-   (1) The first reagent (as buffer solution):    -   Chelating reagent: none    -   Organic solvent: none    -   Stabilizer (dispersant: nonionic surfactant):        -   TritonX-100® (polyoxyethylene octylphenyl ether): 1.0% by            weight    -   Making reagent: triethanolamine 10 mM        Into a mixture of above components, 7% by weight of ammonium        chloride was added to adjust pH to pH 10 and purified water was        added up to the total volume of 1 liter, which was stored in a        general-purpose container.-   (2) The second reagent (as coloring reagent solution)    -   Chelating reagent: F28 tetraphenylporphyrin: 0.5 g/L    -   Organic solvent: Dimethylsulfoxide (DMSO): 20% by weight    -   Stabilizer: TritonX-100® (polyoxyethylene octylphenyl ether)        1.0% by weight    -   Masking reagent:        -   triethanolamine: 10 mM        -   ethylenediamine tetraacetic acid (EDTA 2K) (Dojin Chemical):        -   0.1 mM            Into a mixture of above components, 0.05M of MOPS (buffer)            was added to adjust pH to pH 7.0. Then, the total volume was            increased to 1 liter with purified water and the resulting            solution was stored in a general-purpose storing container.

The concentration of lithium in the control serum sample was determinedquantitatively by the same procedure as Example 1 by using lithiumreagent compositions prepared in Example 8 and Example 9. Results aresummarized in [Table 5] of FIG. 11 “Comparison of measured values amongdifferent masking agents”.

FIG. 11 reveals that measured values coincide over 95% for among ameasured value for triethanolamine alone (0.83 mM), a measured value forethylenediamine tetra acetic acid (EDTA) alone (0.83 mM) and a measuredvalue for their combination use (0.82 mM).

This result shows that almost same measured values can be obtainedregardless of type of masking agent used or their combination.Therefore, suitable masking agent(s) can be used to prevent degradationof the reagent caused by trace metal ions which may be contained in astocked reagent. The masking agent can be used for a test samplecontaining excess inclusion ions.

As explained above Examples according to this invention, theconcentration of lithium in aqueous solution such as environmentalsample and biological specimen can be determined by the convenientcolorimeter and can be judged immediately by visual observation.

A scope of this invention should not be limited to the Examples but isdefined by claims. Details of Examples can be changed, altered andmodified provided that the characteristic of this invention is notimpaired. For example, in Examples 1-9, the reagent composition fordetermining the concentration of lithium is divided into two reagents ofthe first and second reagents separately to store the reagentcomposition for a longer term. However, if measurement is carried outwithin a short period, the first reagent and the second reagent can bemixed from the beginning and the resulting mixture is used in themeasurement.

The invention claimed is:
 1. A lithium reagent composition for measuringthe quantity of lithium in serum or blood plasma, comprising a compoundhaving a structure represented by the formula (I):

in which all hydrogens bonded to carbons of a tetraphenylporphyrin arereplaced by fluorine atoms, a water-miscible organic solvent selectedfrom the group consisting of dimethylsulfoxide (DMS), dimethylformamide(DMF) and dimethylacetamide (DMA), and a pH modifier for adjusting thepH to a range from pH 5 to pH 12, wherein said reagent composition is anaqueous solution.
 2. The lithium reagent composition of claim 1, inwhich said pH modifier is selected from acids including hydrochloricacid, nitric acid, acetic acid, phosphoric acid, citric acid, carbonicacid, bicarbonic acid, oxalic acid and their salts, alkali medicineincluding sodium hydroxide, potassium hydroxide, ammonia and theirsalts.
 3. The lithium reagent composition of claim 1, in which said pHmodifier is pH buffer.
 4. The lithium reagent composition of claim 3, inwhich said pH buffer is selected from citric acid, carbonic acid,bicarbonic acid, phosphoric acid, succinic acid, phthalic acid, ammoniumchloride, sodium hydroxide, potassium hydroxide, MES as Good buffer,Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO,POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO, CAPS and theirsalts.
 5. The lithium reagent composition of claim 3, in which thereagent composition develops a color reaction for lithium in a rangefrom pH 5 to pH
 11. 6. The lithium reagent composition of claim 1,including further a stabilizer.
 7. The lithium reagent composition ofclaim 1, in which said stabilizer is nonionic surfactant and/or anionicsurfactant.
 8. The lithium reagent composition for lithium according toclaim 7, in which said nonionic surfactant is selected from esters ofsorbitan fatty acid, partial esters of pentaerythritol fatty acid,esters of propylene glycol fatty acid, glycerin fatty acid monoester,polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether,polyoxyethylene polyoxypropyleneglycol, partial esters ofpolyoxyethylene fatty acid, partial esters of polyoxyethylene sorbitolfatty acid, esters of polyoxyethylene fatty acid, fatty acid di-ethanolamide, fatty acid monoethanol amide, polyoxyethylene fatty acid amide,polyoxyethylene octylphenyl ether (TritonX-100®), p-nonylphenoxypolyglycidol and their salts.
 9. The lithium reagent compositionfor lithium according to claim 7, in which said anionic surfactant isalkyl sulfate ester salt, polyoxyethylene alkyl ether sulfate salt,alkylbenzenesulfonate salts and alkanesulfonat.
 10. The lithium reagentcomposition from lithium according to claim 9, wherein the anionicsurfactant is sodium dodecyl sulfate, sodium dodecyl benzene sulfonate,sodium polyoxyethylene alkly phenyl ether sulfate or salts thereof. 11.The lithium reagent composition for lithium according to claim 1,including further a masking reagent.
 12. The lithium reagent compositionfor lithium according to claim 11, in which said masking reagent ischosen from triethanolamine, ethylenediamine,N,N,N′,N′-tetrakis(2-pyridylmethl)ethylenediamine (TPEN), pyridine,2,2-bipyridine, propylenediamine, diethyl enetriamine,diethylenetriamine-N,N,N,N″,N″-pentaacetate (DTPA),triethylenetetramine, triethylenetetramine-N,N,N′,N″,N″,N″-hexaacetate(TTHA), 1,10-phenanthroline, ethylenediamine tetraacetate (EDTA),O,O′-bis(2-aminophenyl)ethyleneglycol-N,N,N,N-tetraacetate (BAPTA),N,N-bis(hydroxyethyl)glycine (Bicine),trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate (CyDTA),O,O′-bis(2-aminoethyl)ethyleneglycol-N,N′,N-tetraacetate (EGTA),N-(2-hydroxyl)iminodiacetate (HIDA), imino diacetic acid (IDA), nitriletriacetic acid (NTA), nitrylo tris-methylphosphonate (NTPO) and theirsalts.
 13. The lithium reagent composition for lithium according toclaim 1, in which the coloring of the lithium complex is measured by thesensitivity in the spectrum at a wavelength of 570 nm.
 14. The lithiumreagent composition of claim 1, wherein a coloring is measured at awavelength of 530 nm to 560 nm.
 15. The lithium reagent composition ofclaim 14, wherein the wavelength range is 550 nm.
 16. The lithiumreagent composition for lithium according to claim 1, in which thecoloring of the lithium complex is measured by the sensitivity in thespectrum in a wavelength range from 565 nm to 650 nm.
 17. A lithiumreagent kit comprising a first reagent comprising the stabilizer and thepH modifier defined in claim 1, and a second reagent including thecompound having the structure represented by the formula (I):

in which all hydrogens bonded to carbons of a tetraphenylporphyrin arereplaced by fluorine atoms, the water-soluble organic solvent, selectedfrom the group consisting of dimethylsulfoxide (DMSO), dimethylformamide(DMF) and dimethylacetamide (DMA), said first and second reagents beingstored separately and mixed just before measurement operation to form alithium reagent composition, wherein said lithium reagent composition isan aqueous solution.
 18. The lithium reagent kit according to claim 17,a coloring is measured at a wavelength of 530 nm to 560 nm.
 19. Thelithium reagent kit according to claim 18, wherein the wavelength rangeis 550 nm.
 20. A method for preparing a lithium reagent composition,comprising: dissolving a compound having a structure represented by theformula (I):

in which all hydrogens bonded to carbons of a tetraphenylporphyrin arereplaced by fluorine atoms in a water-miscible organic solvent selectedfrom the group consisting of dimethylsulfoxide (DMS), dimethylformamide(DMF) and dimethylacetamide (DMA), and a pH modifier for adjusting thepH to a range from pH 5 to pH 12 to obtain a mixture, and then addingwater to the mixture.
 21. A lithium reagent for measuring the quantityof lithium in serum or blood plasma, wherein the lithium reagent is adilution of a composition comprising a compound having a structurerepresented by the formula (I):

in which all hydrogens bonded to carbons of a tetraphenylporphyrin arereplaced by fluorine atoms, a water-miscible organic solvent selectedfrom the group consisting of dimethylsulfoxide (DMS), dimethylformamide(DMF) and dimethylacetamide (DMA), and a pH modifier for adjusting thepH to a range from pH 5 to pH 12, wherein the composition is dilutedwith 1L of water to obtain the dilution.
 22. The lithium reagent ofclaim 21, wherein the compound having a structure represented by theformula (I) is present in an amount of 0.5 g/L to 1.0 g/L.